Journal: Cell Death Discovery

Article Title: Interplay between ALK2 R206H mutant receptor and autophagy signaling regulates receptor stability and its chondrogenic functions

doi: 10.1038/s41420-025-02393-0

Figure Lengend Snippet: A Flow cytometry analysis in ATDC5 cells overexpressing ALK2 WT or mutant ALK2 R206H receptors in regular medium (5% FCS) and serum starvation (starvation) conditions. GFP-positive and RFP-positive cells were detected by fluorescence emitted under 488 nm and 561 nm excitation lasers, respectively. Representative histograms of a single experiment with median fluorescence intensity of GFP and RFP are shown. Cumulative plots of six independent experiments show mean ± SD of GFP/RFP fluorescence ratio in serum starvation conditions versus regular medium (DMEM F12 5% FBS). Statistical analysis: One-way ANOVA (Tukey’s multiple comparison) test was performed (ns = not significant; ** P ≤ 0.01; **** P ≤ 0.0001) ( B ) Representative immunoblotting and relative densitometric analysis ( n = 3) on total protein extracts derived from ATDC5 cells grown in regular medium (5% FBS) or in serum starvation condition (starvation). Tubulin and HSP90 were used as loading control. Statistical analysis: unpaired t -test (ns = not significant; ** P ≤ 0.01; **** P ≤ 0.0001) ( C ) Confocal quantification of the number of autophagic vesicles in ATDC5 cells in starvation condition after loading with 50 µM MDC. Data analysis of MDC spot number was performed using the Harmony software (ver 4.5) of the Opera Phenix high-content system. Scale bar, 10 μm. Statistical analysis: unpaired t- test (**** P ≤ 0.0001).

Article Snippet: For immunoblotting, 20–30 μg of protein extract were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted on nitrocellulose membrane, and stained with the specific primary antibodies: anti-Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E,Cell Signaling Technology), anti-p62 (SQSTM1) (PM045; MBL International), anti-GAPDH (D16H11) (Cell Signaling Technology), anti-LC3B (Sigma–Aldrich, L7543), anti-NanoLuc (Promega Corporation, N700A), anti-DDK (FLAG) (Origene, TA50011-100), anti ALK2/ACVR1 (SinoBiological.

Techniques: Flow Cytometry, Mutagenesis, Fluorescence, Comparison, Western Blot, Derivative Assay, Control, Software

Journal: Cell Death Discovery

Article Title: Interplay between ALK2 R206H mutant receptor and autophagy signaling regulates receptor stability and its chondrogenic functions

doi: 10.1038/s41420-025-02393-0

Figure Lengend Snippet: A Histograms show the mRNA expression for the indicated genes expressed as fold increase (mean ± SD) from three independent experiments in ATDC5 cells overexpressing ALK2 WT or mutant ALK2 R206H in normoxic (21% O 2 ) and hypoxic conditions (1% O 2 ,16 h). GAPDH mRNA was used to normalize data. B Immunoblotting and corresponding densitometric analysis with the indicated antibodies of ATDC5 cells overexpressing ALK2 WT or mutant ALK2 R206H in normoxic (21% O 2 ) and hypoxic conditions (1% O 2 ,16 h) ( C ) Histograms show the mRNA expression for the indicated gene expressed as fold increase (mean ± SD) from three independent experiments in ATDC5 cells overexpressing ALK2 WT or mutant ALK2 R206H in normoxic (21% O 2 ) and hypoxic conditions (1% O 2 ,16 h). GAPDH mRNA was used to normalize data. D , E Immunoblotting and relative densitometric analysis with the indicated antibodies of U2OS cells overexpressing ALK2 WT or mutant ALK2 R206H DDK tagged, in normoxic (21% O 2 ) and hypoxic conditions (1% O 2 ,16 h) and treated, as indicated, with 20 μM CQ (2 h). F Confocal microscopy analysis using anti-DDK (red) in U2OS cells expressing ALK2 WT or mutant ALK2 R206H DDK tagged receptors treated or not, as indicated, with chloroquine (CQ, 20 μM, 2 h) in normoxic (21% O 2 ) and hypoxic conditions (1% O 2 ,16 h). DNA was counterstained with DAPI (blue). Scale bar 10 µm. DDK fluorescence intensity was measured by using Fiji software (ImageJ). At least 50 cells from 3 independent experiments were analyzed unpaired t -test (Welch’s correction) was performed. Data are presented as mean ± SD of at least three independent experiments. Symbols (dots) represent individual cells. Statistical analysis: unpaired (ns = not significant; * P ≤ 0.05; **P ≤ 0.01; *** P < 0.001 ****P ≤ 0.0001).

Article Snippet: For immunoblotting, 20–30 μg of protein extract were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted on nitrocellulose membrane, and stained with the specific primary antibodies: anti-Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E,Cell Signaling Technology), anti-p62 (SQSTM1) (PM045; MBL International), anti-GAPDH (D16H11) (Cell Signaling Technology), anti-LC3B (Sigma–Aldrich, L7543), anti-NanoLuc (Promega Corporation, N700A), anti-DDK (FLAG) (Origene, TA50011-100), anti ALK2/ACVR1 (SinoBiological.

Techniques: Expressing, Mutagenesis, Western Blot, Confocal Microscopy, Fluorescence, Software

Journal: Cell Death Discovery

Article Title: Interplay between ALK2 R206H mutant receptor and autophagy signaling regulates receptor stability and its chondrogenic functions

doi: 10.1038/s41420-025-02393-0

Figure Lengend Snippet: A Representative images and relative quantification of EGFP-LC3-labeled autophagosomes colocalization with ALK2-DDK receptor. U2OS EGFP-LC3 cells expressing ALK2 WT or mutant ALK2 R206H DDK tagged receptors were treated with chloroquine (CQ, 20 μM, 2 h) in hypoxic conditions (1% O 2 ,16 h). Images were acquired by spinning disk confocal microscopy and analyzed for colocalization of EGFP-LC3 (green) and DDK (red) dots using the ComeDet plugin of the Fiji ImageJ software. Data are presented as mean ± SD and normalized on total EGFP-LC3 dots. DNA was counterstained with DAPI (blue). Insets show a 3-fold enlargement of the boxed areas. Scale bar, 10 μm is shown. At least 50 cells from 3 independent experiments were analyzed unpaired t -test (Welch’s correction) was performed. (ns = not significant; * P ≤ 0.05; ** P ≤ 0.01; *** P < 0.001 **** P ≤ 0.0001). B Immunoblotting and relative densitometric analysis, numbers report the densitometric values of band intensity, with the indicated antibodies of U2OS cells overexpressing ALK2 WT or mutant ALK2 R206H in normoxic (21% O 2 ) and hypoxic conditions (1% O 2 ,16 h) and stably infected with lentiviral construct expressing shATG5 RNA interference or expressing an empty vector as control (shCTRL).

Article Snippet: For immunoblotting, 20–30 μg of protein extract were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted on nitrocellulose membrane, and stained with the specific primary antibodies: anti-Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E,Cell Signaling Technology), anti-p62 (SQSTM1) (PM045; MBL International), anti-GAPDH (D16H11) (Cell Signaling Technology), anti-LC3B (Sigma–Aldrich, L7543), anti-NanoLuc (Promega Corporation, N700A), anti-DDK (FLAG) (Origene, TA50011-100), anti ALK2/ACVR1 (SinoBiological.

Techniques: Labeling, Expressing, Mutagenesis, Confocal Microscopy, Software, Western Blot, Stable Transfection, Infection, Construct, Plasmid Preparation, Control

Journal: Cell Death Discovery

Article Title: Interplay between ALK2 R206H mutant receptor and autophagy signaling regulates receptor stability and its chondrogenic functions

doi: 10.1038/s41420-025-02393-0

Figure Lengend Snippet: A Immunoblotting and relative densitometric analysis with the indicated antibodies of ALK2 R206H -DDK U2OS cells treated or not, at different time points, with activin A (100 ng/ml) and Rapamycin (Rapa, 100 ng/ml) in serum starvation conditions. Histograms show the mean ± SD of at least three independent experiments. Statistical analysis: unpaired t -test (ns = not significant; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001 **** P ≤ 0.0001). B Representative fluorescence images of U2OS cells expressing mutant ALK2 R206H DDK tagged receptors treated or not, as indicated, with Rapamycin (Rapa, 100 ng/ml, 5 h) and/or chloroquine (CQ, 20 μM, 2 h), immunostained with anti-LAMP2 (red) and anti-DDK (green) antibodies and counterstained with DNA (DAPI, blue). Scale bar 10 µm. Images were acquired by spinning disk confocal microscopy and analyzed for DDK fluorescence intensity using the Fiji ImageJ software. Data are presented as median with interquartile range. N ≥ 20 cells per sample from two independent experiments. Symbols represent individual cells. Insets show a 3-fold enlargement of the boxed areas. Statistical analysis: one-way ANOVA (Tukey’s multiple comparison) test was performed (** P ≤ 0.01; **** P ≤ 0.0001). C Flow cytometry analysis of ALK2 R206H ATDC5 cells cultured in serum starvation conditions, left untreated (NT) or treated with activin A (100 ng /ml) alone (DMSO), or in combination with Chloroquine (CQ, 20 µM, 2 h), Rapamycin (Rapa, 100 ng/ml, 24 h) for 24 h. Representative histograms show the median fluorescence intensity (MFI) of fluorescence GFP and RFP emitted as a result of excitation with blue (488 nm) and yellow (561 nm) lasers, respectively. Cumulative plots report the mean ± SD of GFP/RFP fluorescence ratio versus non-treated (NT) cells ( n = 3). Statistical analysis: one-way ANOVA (Tukey’s multiple comparison) test was performed (ns = not significant; ** P ≤ 0.01).

Article Snippet: For immunoblotting, 20–30 μg of protein extract were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted on nitrocellulose membrane, and stained with the specific primary antibodies: anti-Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E,Cell Signaling Technology), anti-p62 (SQSTM1) (PM045; MBL International), anti-GAPDH (D16H11) (Cell Signaling Technology), anti-LC3B (Sigma–Aldrich, L7543), anti-NanoLuc (Promega Corporation, N700A), anti-DDK (FLAG) (Origene, TA50011-100), anti ALK2/ACVR1 (SinoBiological.

Techniques: Western Blot, Fluorescence, Expressing, Mutagenesis, Confocal Microscopy, Software, Comparison, Flow Cytometry, Cell Culture

Journal: Cell Death Discovery

Article Title: Interplay between ALK2 R206H mutant receptor and autophagy signaling regulates receptor stability and its chondrogenic functions

doi: 10.1038/s41420-025-02393-0

Figure Lengend Snippet: A Chondrogenic differentiation micromass assay in ATDC5 ALK2 R206H cells and in ATDC5 ALK2 WT cells. Cells were incubated for 21 days in differentiation medium containing or not (NT), activin A (100 ng/ml) and treated, as indicated, with autophagy inhibitors Chloroquine (CQ, 20 μM), 3-Methyladenine (3-MA, 20 μM) or autophagy inducers Rapamycin (Rapa, 100 ng/ml), Spermidine (SPD, 20 μM) or DMSO. Representative images of Alcian blue staining. Histograms representing Alcian blue quantification measured by absorbance at 595 nm after solubilization with guanidine hydrochloride. All results represented the mean ± SD of at least three independent experiments. Statistical analysis: One-way ANOVA (Tukey’s multiple comparison) test was performed (** P ≤ 0.01; *** P < 0.001 **** P ≤ 0.0001). B Real-time PCR of Col10a1 mRNA in ATDC5 cells treated or not with activin A for 21 days in micromass cultures. mRplp0 was used to normalize data. All results represented the mean ± SD of at least three independent experiments. Statistical analysis: unpaired t- test (** P ≤ 0.01; *** P < 0.001 **** P ≤ 0.0001). C Histogram showing measurement of ALP activity in ATDC5 cells treated or not as indicated for 5 days in micromass culture. 5 × 10 4 cells were homogenized in 1 ml of assay Buffer, diluted 1:10 in assay Buffer, and 80 μl was used to measure ALP activity. Assays were performed following the fluorimetric kit protocol (MAK411, Sigma–Aldrich) and measured at O.D. 405 nm reading with Varioskan LUX Plate Reader. All results represented the mean ± SD of at least three independent experiments. Statistical analysis: A one-way ANOVA (Tukey’s multiple comparison) test was performed (** P ≤ 0.01; *** P < 0.001). Chondrogenic differentiation micromass assay in ATDC5 ALK2 R206H cells stable expressing shRNA for ATG4 (shATG4) ( D ) or ATDC5 ALK2 WT and ALK2 R206H cells stable expressing shRNA for Rubicon (shRubicon) ( E ), upon lentiviral infection. A lentiviral empty vector was used as control (shCTRL). Cells were incubated for 21 days in differentiation medium containing or not (NT), activin A (100 ng/ml) and treated as indicated with Rapamycin (Rapa 100 ng/ml) or DMSO. Representative images of Alcian blue staining and relative histograms showing Alcian blue quantification measured by absorbance at 595 nm after solubilization with guanidine hydrochloride. All results represented the mean ± SD of at least three independent experiments for ( D ) and from six independent experiments for ( E ). Statistical analysis: One-way ANOVA (Tukey’s multiple comparison) test was performed (* P ≤ 0.05; ** P ≤ 0.01, **** P ≤ 0.0001).

Article Snippet: For immunoblotting, 20–30 μg of protein extract were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted on nitrocellulose membrane, and stained with the specific primary antibodies: anti-Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E,Cell Signaling Technology), anti-p62 (SQSTM1) (PM045; MBL International), anti-GAPDH (D16H11) (Cell Signaling Technology), anti-LC3B (Sigma–Aldrich, L7543), anti-NanoLuc (Promega Corporation, N700A), anti-DDK (FLAG) (Origene, TA50011-100), anti ALK2/ACVR1 (SinoBiological.

Techniques: Incubation, Staining, Comparison, Real-time Polymerase Chain Reaction, Activity Assay, Expressing, shRNA, Infection, Plasmid Preparation, Control

Journal: Cell Death Discovery

Article Title: Interplay between ALK2 R206H mutant receptor and autophagy signaling regulates receptor stability and its chondrogenic functions

doi: 10.1038/s41420-025-02393-0

Figure Lengend Snippet: Schematic illustration showing a simplified model of the interplay between the mutant ALK2 R206H receptor (without showing the type I/type II receptors tetramer) and the mTOR/autophagy signaling in the regulation of the balance between receptor degradation and chondrogenesis upon hypoxic conditions or activin A stimulation.

Article Snippet: For immunoblotting, 20–30 μg of protein extract were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted on nitrocellulose membrane, and stained with the specific primary antibodies: anti-Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E,Cell Signaling Technology), anti-p62 (SQSTM1) (PM045; MBL International), anti-GAPDH (D16H11) (Cell Signaling Technology), anti-LC3B (Sigma–Aldrich, L7543), anti-NanoLuc (Promega Corporation, N700A), anti-DDK (FLAG) (Origene, TA50011-100), anti ALK2/ACVR1 (SinoBiological.

Techniques: Mutagenesis

Journal: Biology

Article Title: BMP-2-Driven Osteogenesis: A Comparative Analysis of Porcine BMSCs and ASCs and the Role of TGF-β and FGF Signaling.

doi: 10.3390/biology14060610

Figure Lengend Snippet: Figure 6. Receptor expressions of ALK2, ALK4, ALK6, ALK5, ALK7, BMPR-II in pASC. Grouped representation of the respective receptor expression in the course of osteogenic differentiation (OM +/−BMP-2). From day 19, there is a significant induction of ALK 2, ALK 6, and ALK 5 with the addition of BMP-2. BMPR-II expression in the OM group decreased in OM and tended to stay increased under BMP-2 supplementation from day 19, but was not considered significant (* p ≤0.05, ** p ≤0.01; n = 6, BMP-2 450 ng/mL).

Article Snippet: The respective conjugated antibodies were used for the expressions of ALK3 (Cat. No.: AF436), ALK 5 (Cat. No.: FAB5871), ALK6 (Cat. No.: FAB5051A), TGF-β2-RII (Cat. No.: FAB532P), ALK7 (Cat. No.: FAB77491A), ALK2 (Cat. No.: AF637), ALK4 (Cat. No.: MAB2221), and BMPR-II (Cat. No.: AF811) (by R&D Systems, Minneapolis, MN, USA), and the pASCs and pBMSCs were compared for their expressions of the specific surface antigens CD45 (Cat. No.: MCA1568GA, BioRad, Hercules, CA, USA), HLA-DR (human leukocyte antigen–antigen D-related surface molecule) (Cat. No.: MCA2314F, Bio-Rad, Hercules, CA, USA), CD29 (Cat. No.: 561,496, BD Pharmingen, Franklin Lakes, NJ, USA), CD79alpha (Bio-Rad, Cat. No.: MCA2538GA), CD14 (Cat. No.: MCA1568GA, Bio-Rad, Hercules, CA, USA), CD31 (Cat. No.: AF3387, R&D Systems, Minneapolis, MN, USA), CD105 (Cat. No.: NB110-58718APC, Novus Biologicals, Minneapolis, MN, USA), CD26 (, Cat. No.: NB600-552APC, Novus Biologicals, Minneapolis, MN, USA), CD73 (, Cat. No.: AF4488, R&D Systems, Minneapolis, MN, USA), CD90 (Cat. No.: 559,869, BD Pharmingen, Franklin Lakes, NJ, USA), CD34 (Cat. No.: 81289, abcam, Cambridge, UK), and CD44 (Cat. No.: 5531, BD Pharmingen, Franklin Lakes, NJ, USA).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: BMP10 accelerated spinal astrocytic activation in neuropathic pain via ALK2/smad1/5/8 signaling

doi: 10.3389/fphar.2024.1426121

Figure Lengend Snippet: Graphical representation of the experimental design. Experiment 1: Changes in pain sensitization and expressions of BMP10, ALK2, Smad1/5/8, phosphorylated Smad1/5/8, and GFAP after SNI in mice. Experiment 2: The effects of BMP10 siRNA on SNI-induced pain hypersensitivity and astrocytic activation. Experiment 3: The involvements of ALK2 in BMP10-induced pain hypersensitivity and astrocytic activation. Experiment 4: The effects of Smad1 siRNA on BMP10-induced pain hypersensitivity and astrocytic activation.

Article Snippet: The membranes were incubated with appropriate antibodies, including mouse anti-GFAP antibody (1:1,000, ab279289, Abcam), mouse anti-BMP10 antibody (1:1,000, 462732, Novus Biologicals, CO, United States), rabbit anti-ALK2 antibody (1:100, PA5-114818, Thermo Fisher Scientific, MA, United States), rabbit anti-phospho-Smad1/5/8 (1:1,000, AB3848-I, Sigma Aldrich, Germany), rabbit anti-Smad1/5/8 (1:1,000, NB100-56656, Novus Biologicals, CO, United States) and mouse anti-β-actin (1:1,000, AF2815, Beyotime Biotechnology , Shanghai, China) overnight at 4°C.

Techniques: Activation Assay

Journal: Frontiers in Pharmacology

Article Title: BMP10 accelerated spinal astrocytic activation in neuropathic pain via ALK2/smad1/5/8 signaling

doi: 10.3389/fphar.2024.1426121

Figure Lengend Snippet: Expression levels of ALK2, Samd1/5/8, and p-Smad1/5/8 in mouse ipsilateral spinal dorsal horn after SNI. (A–C) Western blot analysis showed the expression levels of ALK2, Samd1/5/8, and p-Smad1/5/8 in the ipsilateral (above) and contralateral (below) spinal dorsal horn of sham and SNI mice; (D) Double immunofluorescence staining showed the coexpression of ALK2 (red) with GFAP, Iba-1 and NeuN (green) in the ipsilateral spinal dorsal horn of SNI mice on postoperative Day 14 (scale bar = 50 μm/25 μm). Data are presented as mean and SEM; sham mice versus SNI mice on postoperative Days 7 and 14, *** p < 0.001 (n = 6).

Article Snippet: The membranes were incubated with appropriate antibodies, including mouse anti-GFAP antibody (1:1,000, ab279289, Abcam), mouse anti-BMP10 antibody (1:1,000, 462732, Novus Biologicals, CO, United States), rabbit anti-ALK2 antibody (1:100, PA5-114818, Thermo Fisher Scientific, MA, United States), rabbit anti-phospho-Smad1/5/8 (1:1,000, AB3848-I, Sigma Aldrich, Germany), rabbit anti-Smad1/5/8 (1:1,000, NB100-56656, Novus Biologicals, CO, United States) and mouse anti-β-actin (1:1,000, AF2815, Beyotime Biotechnology , Shanghai, China) overnight at 4°C.

Techniques: Expressing, Western Blot, Double Immunofluorescence Staining

Journal: Frontiers in Pharmacology

Article Title: BMP10 accelerated spinal astrocytic activation in neuropathic pain via ALK2/smad1/5/8 signaling

doi: 10.3389/fphar.2024.1426121

Figure Lengend Snippet: ALK2 was involved in BMP10-induced pain hypersensitivity and astrocytic activation. (A, B) The time course of PWT and TWL in normal mice after intrathecal delivery of BMP10 peptide and reagents; Data are presented as mean and SEM, Vehicle group versus BMP10 group * p < 0.05, ** p < 0.01, *** p < 0.001; BMP10+ALK2-IN-2 group versus BMP group # p < 0.05, ## p < 0.01, ### p < 0.001 (n = 12); (C) Representative movement traces in OFT tests of mice after intrathecal delivery of BMP10 peptide and reagents; (D–F) Time in center zone, average speed and total distance of mice in open field after intrathecal delivery of BMP10 peptide and reagents; (G, H) immunofluorescence staining showed the expression of GFAP in the spinal dorsal horn after intrathecal delivery of BMP10 peptide and reagents (scale bar = 200/50 μm); (I) Western blot showed the expression of GFAP in the spinal cord after intrathecal delivery of BMP10 peptide and reagents; Data are presented as mean and SEM, Vehicle group versus BMP10 group * p < 0.05, ** p < 0.01, *** p < 0.001; BMP10+ ALK2-IN-2 group versus BMP group # p < 0.05, ## p < 0.01, ### p < 0.001 (n = 6).

Article Snippet: The membranes were incubated with appropriate antibodies, including mouse anti-GFAP antibody (1:1,000, ab279289, Abcam), mouse anti-BMP10 antibody (1:1,000, 462732, Novus Biologicals, CO, United States), rabbit anti-ALK2 antibody (1:100, PA5-114818, Thermo Fisher Scientific, MA, United States), rabbit anti-phospho-Smad1/5/8 (1:1,000, AB3848-I, Sigma Aldrich, Germany), rabbit anti-Smad1/5/8 (1:1,000, NB100-56656, Novus Biologicals, CO, United States) and mouse anti-β-actin (1:1,000, AF2815, Beyotime Biotechnology , Shanghai, China) overnight at 4°C.

Techniques: Activation Assay, Immunofluorescence, Staining, Expressing, Western Blot